Vol. 9(39), pp. 1006-1012, 17 October, 2015 DOI: 10.5897/JMPR2015.5743 Article Number: 26FAE7955993 ISSN 1996-0875 Journal of Medicinal Plants Research Copyright © 2015 Author(s) retain the copyright of this article http://www.academicjournals.org/JMPR
Full Length Research Paper
Extraction of essential oil from inflorescences of Dysphania ambrosioides and its activity against Botrytis cinerea
Juliana Pace Salimena1, Fernando Pereira Monteiro1,2*, Paulo Estevão de Souza1,2 and Jorge Teodoro de Souza2
1Department of Agriculture, Lavras Federal University, 37200-000 Lavras, MG, Brazil. 2Department of Phytopathology, Lavras Federal University. 37200-000 Lavras, MG, Brazil.
Received 17 January, 2015; Accepted 5 October, 2015
Essential oils are natural complex substances biosynthesized by plants, and many of them have antimicrobial properties. Dysphania ambrosioides is a medicinal plant traditionally used as an anthelmintic medicine. In this study, the antifungal activity of D. ambrosioides essential oil was tested against Botrytis cinerea, which is responsible for large economic losses in the post-harvest of roses. Inflorescences of D. ambrosioides yielded 1.3 mg.g-1 of essential oil in fresh material, corresponding to a content of 0.13%. Gas chromatography coupled with mass spectrometry (GC-MS) revealed 11 compounds in the essential oil with ascaridol and O-cymene corresponding to 80% of the total. The essential oil reduced B. cinerea mycelial growth by approximately 60% and spores germination by 51% at the concentration of 1000 ppm. However, no apparent morphological changes were observed in scanning electron microscopy analyzes. The essential oil was not able to reduce mycelial growth on rose petals and caused a color change in the petals 24 h after the treatment. Although the essential oil has little potential to control B. cinerea on roses due to the color change it causes, its activity on mycelial growth and spores germination could be exploited in other pathosystems.
Key words: Rosa hybrida, grey mold, chenopodium ambrosioides.
INTRODUCTION
Essential oils are natural plant products also known as vapor phase, suggesting their use as fumigants volatile oils. They are fragrant substances with an oily (Umpiérrez et al., 2012). The use of fumigants avoids the consistency composed of a complex mixture of volatile direct contact with plants protecting them from possible molecules that may have multiple antimicrobial properties harmful effects and ensures more security to the (Bassolé and Juliani, 2012). Some essential oils have consumers (Shao et al., 2013; Umpiérrez et al., 2012). activity not only by direct contact but also through the Dysphania ambrosioides (Chenopodiaceae sin.
*Corresponding author. E-mail: [email protected].
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Salimena et al 1007
Chenopodium ambrosioides) is a medicinal plant mainly MATERIALS AND METHODS used in folk medicine against intestinal parasites since D. ambrosioides cultivation immemorial times. The plant is native to Central and South America and has an annual or perennial cycle Dysphania ambrosioides was grown in tissue culture with standard depending on the natural variety. This herb has a marked procedures (Gangopadhyay et al., 2002). Five seedlings of odor and is popularly known as worm seed, mastruz, approximately 5 mm long were inserted in a glass bottle containing goosefoot, paico or epazote (Lorenzi and Matos, 2002; MS basal medium (Murashige and Skoog, 1962) without hormone addition. The bottles remained for 40 days in a growth chamber Ortega-Ramirez et al., 2014). D. ambrosioides essential with a photoperiod of 16 h of light and 8 h of darkness and oil has toxic effects against fungi (Cabral et al., 2013), temperature between 25 and 28°C. After this incubation period, the bacteria (Liu et al., 2013) and nematodes (Salifou et al., seedlings were transferred to a greenhouse and were acclimated 2013). for two weeks. After the acclimation, plants were transferred to The major compounds found in the essential oil of D. boxes measuring 16 m long x 1.4 m wide x 1.1 m high filled with ambrosioides are p-cymene, carvacrol, isoascaridole and coarse sand and irrigated with a nutrient solution adjusted to pH 5.7±0.1. The composition of the nutrient solution used to fertilize D. ascaridoles (Monzote et al., 2014; Ávila-Blanco et al., ambrosioides was calcium nitrate (760 mg/L), ammonium sulfate 2014). The ascaridoles are mainly responsible for its (255 mg/L), monoammonium phosphate (110 mg/L), potassium activity against fungi, including plant pathogens (Jardim chloride (400 mg/L), magnesium sulphate (375 mg/L), FeEDDHMA et al., 2008; Zabka et al., 2009; Nisar et al., 2013). The (44 mg/L), manganese sulphate (6.7 mg/L), boric acid (3.28 mg/L), development of natural fungicides with ascaridoles is zinc sulphate (2.96 mg/L), copper sulphate (0.36 mg/L) and sodium molybdate (0.05 mg/L). Plants were cultivated for 60 days in the encouraging due to its broad range of activity against sand box and harvested at flowering in 20 May, 2014, at 9 am in a several plant pathogens (Jardim et al., 2008). Other mild day with no rainfall. The voucher specimen is deposited in the compounds in the oil may play a significant role in Biology Department herbarium of Lavras Federal University under disease control because the complete essential oil has accession number 10137. better activity in comparison with its pure major compounds (Monzote et al., 2014). Essential oil extraction and chemical composition The essential oil of D. ambrosioides may be used as a natural fungicide in postharvest protection of agricultural Essential oil extraction was performed by steam distillation (Marconi commodities, such as cut flowers (Jardim et al., 2008). MA480). One hundred grams of fresh inflorescence was used for extraction. The hidrolact was collected after 90 min, and the The distances between the cut flower production areas essential oil was purified by liquid-liquid partition with 25 ml and consumer market may be long. For example, roses dichloromethane for three times. Organic fractions were combined (Rosa hybrida) from South American countries are and dried with anhydrous magnesium sulfate. The salt was transported to the USA or Europe, while roses from removed by simple filtration and solvent was evaporated at room Kenya and Ethiopia are transported via Western Europe temperature in a gas exhaust chapel until constant weight. After extraction of the essential oil each flask was transferred to to Eastern Europe and Asian countries (Harkema et al., amber glass bottles with stoppers and screw caps and weighed 2013). Even at low transportation temperatures the roses with an analytical balance (Marte Científica, Model AL500). may be damaged by fungi (Tuset, 1987). Essential oil content was calculated with the following formula: The rose trade has great economic importance to Content (%) = (essential oil mass (g) x 100)/ fresh plant material Brazil. However, its competitiveness in the world market weight (g). The analysis of chemical composition was done in is greatly affected by the lack of research and triplicate. The quantitative analyzes were performed by chromatography coupled with a flame ionization detector hydrogen improvement in areas such as resistance to pathogens (GC-FID) and Agilent® 7890A system equipped with a fused silica (Barbieri and Stumpf, 2005). Roses are specially harmed capillary column gas-phase HP-5 (30 m long × 0.25 mm inside by Botrytis cinerea (synonym: Botryotinia fuckeliana) that diameter x 0.25 mm thick film). may attack more than 200 host species worldwide. Many Helium was used as carrier at a flow of 1.0 ml/min. Injection and fungicides have failed to control this pathogen due to its detection temperatures were maintained at 220°C and 240°C, genetic plasticity (Williamson et al., 2007). respectively. Initial oven temperature was 40°C followed by a temperature ramp of 3°C/min up to 200°C and 10°C/min up to Excessive spraying of conventional chemicals used to 250°C. The essential oil was diluted with 1% ethyl acetate and 1µL control pests may harm the environment. Opposite to this injected into the chromatograph using the split mode at 1:50. view, there is a search for environmentally safe Quantitative analysis was obtained by integrating the total ion alternatives (Verma et al., 2009). Farmers have been chromatogram and the content of the eluted constituents expressed using a variety of strategies against plant pathogens as percentage of peak relative area. Qualitative analyzes were done by gas chromatography coupled (Dominiak and Ekman, 2013). In addition, plant-derived to mass spectrometry (GC-MS) with an Agilent® 5975C. This is compounds such as essential oils could also be applied. operated in scan mode by electronic impact ionization at 70 eV at The aims of this study were to determine the chemical the speed of 1.0 scan/s and mass acquisition interval of 40 to 400 composition and yield of the essential oil extracted from m/z. The chromatographic conditions were identical to the ones inflorescences of D. ambrosioides, and test its activity used in the quantitative analyzes. The components were identified against B. cinerea pathogenic to roses. No previous by comparing their calculated Kovats retention indices (IKc) with retention indices (IK) reported in the literature (Adams, 2007; reports were found on the use of D. ambrosioides Kováts, 1965) and by mass spectral comparisons. IKc were essential oil in the control of rose diseases. calculated by applying the equation of Van Den Dool and Kratz 1008 J. Med. Plants Res.
(1963) on the basis of standard n-alkanes co-injection C8- measured every 24 h up to 144 h. C20 (Sigma Chemical Co.).
Statistical analyses Effects of the essential oil on B. cinerea All statistical analyses were done with the R software (R Core B. cinerea was obtained from the CML mycological collection Team, 2014). Comparisons among means were performed using (Lavras Federal University, accession number 2317). Spores were Tukey's test at 5% probability. produced on PDA medium at 25°C for eight days and suspensions 5 were adjusted to 2x10 spores/ml. Spore suspensions with this concentration were used in all experiments described below. RESULTS AND DISCUSSION Petri dishes divided in two sections were used to assess mycelial growth after treatment with the essential oil. On the center of one section containing 10 ml of PDA, an aliquot of 2 µL of the conidial Essential oil content and chemical composition suspension was added. In the other section, a cotton plug of approximately 0.16 g moistened with 1ml of a mixture of essential The yield and content of essential oil from D. oil at 250, 500, 750 and 1000 ppm dissolved in 3% water- ambrosioides inflorescences was 1.3±0.56 mg.g-1 and polysorbate (Tween 80) was placed. These cotton plugs were used to allow volatile dissipation for a long period of time. The controls 0.13% in fresh material, respectively. In this study, the were sterile distilled water-polysorbate at 3% in one section and the essential oil was extracted exclusively from fungal spores in the other. The mycelial radial diameter was inflorescences of D. ambrosioides. Other authors found measured every 24h up to 96h. Mycelial growth rate was calculated essential oil contents varying from 0.3 to 1.8% when the according to Oliveira (1991): MGR=∑(D-Dp)/N where -1 whole aerial part was employed (Navaei and Mirza, 2004; MGR=mycelial growth rate (cm.day ), D=current average diameter Rondelli et al., 2012). These differences in oil content (cm), Dp=previous day average diameter (cm), and N=number of days after spore deposition. All experiments were installed in a may be explained by water stress, growing season and completely randomized design with three replicates. stage of development at harvest, which may alter the The effect on spore germination was assessed by using Petri chemical composition and yield (Morais, 2009). plates divided in two sections. In one section containing 10 ml of Inflorescences appear to yield lower amounts of 2% water-agar, 2 ml of the conidial suspension described above essential oil when compared to seeds and fruits that was added onto the medium surface. In the other section, a cotton contain most of essential oil of the plant (Dembitsky et al., plug moistened with 1 ml of a solution composed of 1% essential oil and 3% water-polysorbate was added. Control plugs were treated 2008). In fact, observations done in another study with 1 ml of water-polysorbate. Spores germination was evaluated indicate that the yield of essential oil is approximately 9 every 24 h up to 96 h. Each treatment was assessed by adding 100 times higher in fruits and seeds than in inflorescences µL onto microscope slides and four randomized fields were (Unpublished data). The 11 identified chemical assessed. Counting was repeated five times per treatment. The first constituents represented 98.2% of the essential oil 100 randomly selected conidia were counted and discriminated in germinated or non-germinated. analyzed (Table 1). Scanning electron microscopy analyses were done on cultures The identity of the essential oil components was similar grown for 3 days at 25°C in the presence of 1% essential oil either to those found in the literature, with exception of allyl on cotton plugs as described above or with 100 µL applied directly hexanoate and cyclohex-2-enone,4-hydroxy-4-methyl onto the surface of the medium. Observations on possible which were found for the first time in the essential oil alterations in the mycelium and conidia as affected by the essential extracted from D. ambrosioides. The relative amounts of oil were performed. Mycelial discs measuring 9 mm were collected from the plates, inserted in tubes with 1.5 ml of Karnovsky's fixative the components in the essential oil differed from the and stored at 4°C. After 24 h, each treatment was washed three values found by other authors. The major components in times for 10 min in 0.05M cacodylate buffer solution, transferred to inflorescences were ascaridole and O-cymene, a 1% osmium tetroxide solution for 1h and washed with distilled corresponding to approximately 80% of the total (Table water three times followed by dehydration in an acetone series (25, 1), while the major compounds in leaves were α- 50, 70, 90 and 100%). After dehydration, samples were submitted terpinene and O-cymene (Tapondjou, 2002; Singh et al., to the critical point dryer (Balzers CPD 030) to replace acetone by 2008). Comparatively, the essential oil extracted from CO2. Specimens were placed on stubs covered with aluminum paper inflorescences has more ascaridole than its precursor α- and carbon double-sided tape. Gold coating was done using a terpinene and may be more active against plant sputter coater (Balzers SCD 0.50) for observation with a scanning pathogens. electron microscope LEO EVO 40 (Carl Zeiss AG). The images were recorded at 20 kV accelerating voltage and 10 mm of working distance. Volatile effect on mycelial growth and spore germination Effect on mycelial growth on rose petals There was a significant inhibition in mycelial growth of B. A 5 mm diameter mycelial disk of B. cinerea was placed in the cinerea at 1000 ppm (Figure 1). Mycelial growth was center of a detached rose petal. Petals were placed individually into Petri dishes in three replicates. Cotton plugs humidified with 1 ml of inhibited by 59.8% and mycelial growth rate by 52.3% 1% D. ambrosioides essencial oil and controls with 1 ml of sterile when compared to the control at the highest distilled 3% water-polysorbate. Mycelial radial diameter was concentration of essential oil (1000 ppm). Higher Salimena et al 1009
Table 1. Constituents of the essential oil extracted from inflorescences of D. ambrosioides identified by using gas chromatography.
Kovats index Compound Kovats index (Calculated)1 Content (%)3 (Literature)2 α-terpinene 1013.58 1012 4.63 o-cymene 1021.62 1021 22.86 Sylvestrene 1025.13 1027 0.30 Allyl hexanoate 1072.64 1073 0.19 Cyclohex-2-enone,4-hydroxy-4-methyl 1120.58 1122 0.99 Ascaridole 1235.62 1237 57.28
1 Kovats indices calculated by applying the equation of Van Den Dool and Kratz (1963) on the basis of standard n-alkanes co-injection C8- 2 3 C20 (Sigma Chemical Co.); Kovats indices from Pubchem, Pherobase and ChemSpider databases; Percentage of the compound when using a HP-5 column.
Figure 1. Effect of essential oil from inflorescence of D. ambrosioides on mycelial growth of B. cinerea. Black bars represent mycelial growth up to 72 h and grey bars represent mycelial growth rate (cm/day). Error bars represent standard error. Regression equation for mycelial growth considering the intervals between 0 and 1000ppm was estimated as following y=-0.002547x + 5.146667 (R2=78.21).
inhibition was found by Tripathi et al. (2008) who 96 h after the treatment (Figure 2). Germination of spores observed 100% of mycelial growth inhibition at 500 ppm was reduced by 58.3, 48.1 and 48.3% at 48, 72 and 96 h, for B. cinerea. One possible reason for the differences respectively, after the application of the essential oil. The may be the sensitivity of the isolates used in the different ethanolic extract of this plant was reported to inhibit spore studies. Ascaridole, carvacrol and thymol are the only germination by 18% in the closely related species B. components with activity against B. cinerea mentioned in elliptica (Hsieh et al., 2005). There were no morpholo- the literature (Jardim et al., 2008; Antunes and Cavaco, gical differences between spores or mycelium treated 2010). Although the purified components of the essential and untreated with essential oil (Figure 3). No other stu- oil were not tested, they were found in this study. Spores dies were performed with scanning electron microscopy germination was inhibited by essential oil volatiles up to to evaluate the effects of essential oil from
1010 J. Med. Plants Res.
48 h 72 h 96 h
Figure 2. Effect of D. ambrosioides essential oil on B. cinerea spore germination. Black bars represent treatments and grey bars represent control. Error bars represents the standard error.
Figure 3. Scanning electron microscopy of the effect of D. ambrosioides essential oil on B. cinerea. A – B. cinerea grown in plates containing a cotton plug moistened with 1.6% water-polycarbonate (control); B - B. cinerea treated with 1% essential oil; C - B. cinerea with 1.6% water-polycarbonate applied directly on mycelial discs (control); D - 1% essential oil applied directly on mycelial discs. Salimena et al 1011
A B
Figure 4. Effect of D. ambrosioides essential oil on detached rose petals. A - Control without treatment with essential oil; B - Rose petal treated with essential oil.
Figura 3. Início do experimento.
D. ambrosioides on mycelia and spores of B. cinerea. Conflict of interest
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